Molecular analysis of an echovirus 3 strain isolated from an individual concurrently with appearance of islet cell and IA-2 autoantibodies

Abstract

Growing evidence has implicated members of the genus Enterovirus of the family Picornaviridae in the etiology of some cases of type 1 diabetes (T1D). To contribute to an understanding of the molecular determinants underlying this association, we determined the complete nucleotide sequence of a strain of echovirus 3 (E3), Human enterovirus B (HEV-B) species, isolated from an individual who soon after virus isolation developed autoantibodies characteristic of T1D. The individual has remained positive for over 6 years for tyrosine phosphatase-related IA-2 protein autoantibodies and islet cell autoantibodies, indicating an ongoing autoimmune process, although he has not yet developed clinical T1D. The sequence obtained adds weight to the observation that recent enterovirus isolates differ significantly from prototype strains and provides further evidence of a role for recombination in enterovirus evolution. In common with most HEV-B species members, the isolate exhibits 2C and VP1 sequences suggested as triggers of autoimmunity through molecular mimicry. However, comparisons with the E3 prototype strain and previously reported diabetogenic and nondiabetogenic HEV-B strains do not reveal clear candidates for sequence features of PicoBank/DM1/E3 that could be associated with autoantibody appearance. This is the first time a virus strain isolated at the time of commencement of beta-cell damage has been analyzed and is an invaluable addition to enterovirus strains isolated previously at the onset of T1D in the search for specific molecular features which could be associated with diabetes induction.

FIG. 1.

Autoantibody levels, neutralizing antibody titer against the isolated PicoBank/DM1/E3 strain (top), and viral RNA detected using enterovirus-specific RT-PCR in stool samples during the follow-up of the child participating in the DIPP study. The subject turned autoantibody positive (IAA) at the age of 9 months (marked by a vertical arrow). He had two enterovirus RNA-positive stool samples during the follow-up. The virus isolation was done from a stool sample collected at the age of 6.5 months. E3, PicoBank/DM1/E3 strain. The units of autoantibody levels were as follows: ICA (▴), JDF-U; GADA (•), RU; IAA (▵), RU; and IA-2A (□), RU.

FIG. 2.

Phylogenetic trees expressing the relationship between HEV-B members in different regions of the genome. The trees were derived using the N-J method in the ClustalX software and were bootstrapped 1,000 times before being plotted using the NJPLOT software. Bootstrap values (%) are shown on the branches. (a) Amino acid sequence of P1. (b) Nucleotide sequence of 2C plus 3CD.

FIG. 3.

Nucleotide sequence identities across the genome between E3-Morrisey and a range of enteroviruses, including PicoBank/DM1/E3, together with a schematic representation of the enterovirus genome. The enteroviruses selected are the closest relatives of the two E3 strains. E6, E9, and E12 are prototype strains, and E11-Kar/87 is a recent E11 isolate. SVDV, swine vesicular disease virus. A window of 300 nucleotides and steps of 60 nucleotides were used.

FIG. 4.

Phylogenetic tree expressing the relationship between PicoBank/DM1/E3 and other E3 isolates (W178-128/99, 94CF858, mo/Roma98, si/Roma97, 810/85, 2392-82, 1446-81, Morrisey, and MD86-6393) based on nucleotide identity in part of VP1 (nucleotides 2573 to 2874). Bootstrap values (%) are shown on the branches.

FIG. 5.

Molecular relationships in part of the 5′ UTR (nucleotides 150 to 448 in CBV3-Nancy) between previously reported enterovirus sequences from juvenile diabetic patients (10) and potentially diabetogenic E3, E9, and CBV4 isolates, together with corresponding reference strains. The trees were plotted as for Fig. 2. Bootstrap values (%) are shown on the branches.